Promising anti-leukemic effect of Zataria multiflora extract in combination with doxorubicin to combat acute lymphoblastic leukemia cells (Nalm-6) (in vitro and in silico)

One of the heterogeneous hematologic malignancies of the lymphocyte precursors is ALL. ALL has two incidence peaks that were determined in 2–5 years children and 60 years old adults. Cardiotoxicity of chemotherapeutic drugs is one of important side effects which may occur during or after chemotherapy period. The aim of this study was to evaluate the effect of ZME, Dox, and combinations on Nalm-6 cells. In this vein, the cell viability was assessed by Trypan blue and MTT assay. Evaluation of apoptosis was also analyzed by Annexin-V/PI staining. Moreover, the expression of Bax, Bcl-2, Bcl-xl, hTERT, c-Myc, P53, and P21 genes was detected by Real-Time PCR. Molecular docking as an in-silico method was performed for Bcl-2 and Bcl-xl proteins as well. Our achievements indicated that ZME had dose-dependent effect on Nalm-6 cells and ZME synergistically potentiated Dox effect. The expression of Bax, P53 and P21 genes increased although the expression of Bcl-2 genes decreased when cells treated with ZME/ Dox combination. Molecular docking showed the interactions of carvacrol and thymol in the active cavities of BCL2 and BCL-xl. Regarding to present study, ZME could be utilized as a combinatorial and potential drug for leukemic patients, which is under the treatment by Dox due to reducing the chemotherapy drug doses.


ZME potentiated the cytotoxicity effect of DOX. Metabolic activity of treated cells with ZME and
Dox was investigated with the MTT colorimetric method. As shown in Fig. 2a,b, ZME and Dox separately had cytotoxicity effect on Nalm-6 cells and they reduced the metabolic activity of treated Nalm-6 cells. Figure 2c presents the results of combination doses of ZME and Dox in which those are more efficient than individual doses. Outstandingly, the cytotoxic effect of Dox on Nalm-6 cells was potentiated by ZME using the synergistic combination treatments. ZME enhanced the effect of Dox on programmed cell death. Here, 100 µg/mL ZME plus 10 nM Dox as a combination dose besides 100 µg/mL ZME and 10 nM Dox as individual doses were selected for investigation of Nalm-6 cells apoptosis. As shown in Fig. 3, the combination dose had a considerable increase in the percentage of Annexin-V and Annexin-V/PI positive cells. Consequently, a combination of 100 μg/mL ZME and 10 nM Dox induced 43.4% apoptosis, which is more than 23.71% apoptosis caused by 10 nM Dox alone (P < 0.0001).
Inductive effect of ZME/Dox combination on the gene expression. Nalm-6 cells were exposed to 100 μg/mL ZME, 10 nM Dox and a combination of them for 48 h. Thereupon, the expression of target genes was evaluated using quantitative real-time PCR. According to the results shown in Fig. 4, the expression of the Bax gene increased in the combination dose more than the single doses. These results also indicated the significant decreasing rate in Bcl-2 gene expression which was influenced by the combination dose. Furthermore, Bax and Bcl-2 expression ratio presented that the ZME enhances the Dox apoptotic effect on Nalm-6 cells (Fig. 4).
P53 and P21 were chosen as tumor suppressor genes to assess them in Nalm-6 cells treated with 100 μg/mL ZME, 10 nM Dox and combination of them after 48 h. As presented in Fig. 5a,b, the expression of the P53 and P21 genes was increased individually and combination dose of ZME/ Dox increased the gene expression more than single doses of ZME and Dox. As the results are shown in Fig. 5c,d, 100 μg/mL ZME, 10 nM Dox and a combination of them led to reduce the expression of hTERT and c-Myc genes. The combination dose of ZME/ Dox had more effective than ZME and Dox single doses. ZME had no significant effect on PBMCs. PBMCs were selected as a human normal cell to evaluate the effect of various concentrations of ZME. This study assessed the effect of ZME on PBMCs using MTT and flowcytometry assays after 48 h treatment. The results demonstrated that ZME had no significant impact on PBMCs metabolic activity. Moreover, according to Fig. 3 Fig. 6). These interactions illustrates that these components could set the stage for the apoptosis pathway on Nalm-6 cells treatment with ZME and they may confirm the apoptotic effect of ZME on Nalm-6 cells.

Discussion
With the increase of cancer incidence, many researches have been focused on new chemotherapeutic treatments 27 . One of the most useful and effective chemotherapeutic drugs is Dox which is used to treat wide groups of cancers, such as hematologic malignancies, leukemias, lymphomas and solid tumors like breast cancer. Todays, Dox is utilized widely in the chemotherapeutic regimens of cancer patients, even though Dox has a lot of side effects on patients 28,29 . Cardiotoxicity is one of the most important side effects of Dox causing cardiomyopathy, arrhythmia, congestive heart failure and finally heart failure in patients during or after treatment. To decrease the side effects, the dose of Dox should be reduced 30,31 . Nowadays, the cytotoxic effects of plant extracts on malignant cells are considered to use them as an alternative or complementary drug in chemotherapeutic treatment 32 . The plant extracts have low complications and they could be combined with the low doses of chemotherapeutic drugs in treatments to enhance the cytotoxic effect 33 . The results of this study indicated the cytotoxic effect of ZME individual and in combination with Dox on Nalm-6 cell line. Due to chemical composition analysis of Zataria Figure 1. The ZME (a) and DOX (b) effect of various doses on the viability of Nalm-6 cells using trypan blue assay (mean ± SE, n = 3). These graphs present the changes of ZME and DOX apoptotic effect after 24, 48, 72 h. As shown, different concentrations of ZME and DOX had dose-dependent effect, dose and timedependent effect, respectively. Effect of ZME and DOX combination and single doses (c) after 48 h on the viability of Nalm-6 cells using trypan blue exclusion assay (mean ± SE, n = 3). As shown, combination doses had a more significant apoptotic effect than individual doses on Nalm-6 cells (*P < 0.05, **P < 0.01, ***P < 0.001, relative to untreated cells). The combination index (CI) versus fraction effect (FA) curve (d) of ZME and DOX combination treatment. Exposure of Nalm-6 cells with various combinations of ZME and DOX and cell viability values of trypan blue assay was used for CI vs FA. As reported by FA curve, the CI < 1, = 1, and > 1 indicate respectively synergism, additive effect (solid line), and antagonism effect. Combinations of ZME (100 μg/mL) + DOX (10 nM) on Nalm-6 cells demonstrated the most desirable synergism effect among other combinations. Dose-normalized isobologram analysis of ZME and DOX combination. The CI was calculated according to the normalized isobologram equation (e). (Dx)1 and (Dx)2 indicate the individual dose of ZME and DOX required to inhibit a given level of viability index, and (D)1 and (D)2 are the doses of ZME and DOX necessary to produce the same effect in combination, respectively. Antagonism effect is represented by above points of the effect line, whereas the points are below the effect line demonstrate the synergism effect. Three combination points for Nalm-6 cells were below the effect line, so showed synergism effect. www.nature.com/scientificreports/ Figure 2. The effect of ZME (a) and DOX (b) on the metabolic activity of Nalm-6 cells 24, 48, 72 h after treatments. As shown, ZME had dose-dependent effect but DOX not only had dosedependent effect but also had time-dependent effect. The effect of ZME and DOX combination against single doses after 48 h treatment on metabolic activity of Nalm-6 cells (c). The significant effect of drugs combination is visible in four doses (ZME and DOX: 100 + 10, 100 + 20, 200 + 10, 200 + 20) and we use ZME (100 μg/mL) and DOX (10 nM) as a combination dose for our study (*P < 0.05, **P < 0.01, ***P < 0.001, relative to untreated cells). www.nature.com/scientificreports/ Multiflora essential oil using GC/MS, recent studies have shown that the most ZME constituents are thymol, carvacrol, p-cymene and γ-terpinene, respectively [34][35][36] . The present study also evaluated the apoptotic effect of ZME and Dox individually and in combination on Nalm-6 cells. As shown in our results, the viability of Nalm-6 cells was reduced in exposure to ZME, Dox and their viability surprisingly was decreased in the combination doses more than those of individual. The results of Compusyn ( Fig. 1), in addition, illustrated that three combination doses have significant synergistic apoptotic effect of ZME and Dox on the Nalm-6 cells (ZME and Dox: 100 + 10, 100 + 20, 200 + 10). Furthermore, it is clear in our results that ZME concentration doses potentiated low doses of Dox cytotoxicity effects on Nalm-6 cells. Owing to our results, we would rather choose 48 h incubation for combination investigation by flowcytometry. We, moreover, decided to select one dose before the IC50 doses of Dox and ZME for the combination therapy in order to reach the IC50 effect by combination treatment. So, Nalm-6 cells were treated with ZME: 100 µg/mL, Dox: 10 nM and combination of them to investigation the cytotoxic effect against untreated control. It is necessary to be mentioned that this study assessed the apoptotic effect of ZME and the combination dose with Dox (after 48 h incubation) on PBMCs. Due to flowcytometry results shown in Fig. 3, ZME concentration, individual and in combination, has no significant apoptotic effect on normal cells. In this regard, Janitermi el al. according to their MTT results, claimed that ZME has time-and dose-dependent cytotoxic effect on some cancer cell lines (MCF-7, AGS & HeLa), and it decreased the viability and metabolic activity of treated cancer cells. They also reported that ZME had no cytotoxicity effect on fibroblast cells as a normal cell [37][38][39] , and our MTT results, as well, indicate that there was no considerable cytotoxicity effect on PBMCs that were exposed to 100 and 200 µg/mL ZME. Additionally, according to recent studies, the apoptotic effect of ZME components like thymol, carvacrol, and P-Cymen was evaluated on malignant cell lines. Yi Li and colleagues reported that thymol downregulated the Bcl-2 and Bcl-xl expression while it upregulated the P21 expression in bladder cancer cell lines. They also showed that thymol did not have a cytotoxic effect on the urothelial cell line as non-malignant cell 40 . Recent researchers' findings have shown that BCL-2 family proteins could be key regulators to control apoptosis mechanism. Some anti-apoptotic proteins such as BCL-2, BCL-xl and MCL-1 induce apoptosis process by binding to pro-apoptotic proteins. On the other hand, over expression of these anti-apoptotic proteins are breeding the ground for growth of several cancers by preventing apoptosis 41,42 . Fortunately, our flowcytometry and gene expression results illustrated that ZME provoked the apoptotic effect of Dox on Nalm-6 cells. The expression of Bax as we expected, has increased on treated cells with ZME and Dox. The Bcl-2 expression was down-regulated on exposed cells with ZME and Dox. Moreover, hTERT is a central regulator of multiple hallmarks of various tumors, and the c-Myc and hTERT expression upregulate in most of the malignancies, and there is a correlation between them 43 . In this vein, our findings illustrate that the expression of c-Myc and hTERT reduced much more in combination dose of ZME and Dox than individual doses. In another experiment, Punia et al. also revealed that a combination of Dox and Acacetin (a plant derivative) enhanced the apoptotic effect on one type of lung carcinoma cells 44 . The increasing of BCL2 as an anti-apoptotic factor in many cancers could be a promising target to combat malignant cells. Since BCL2 inhibitors could occupy BH2 and BH3 positions and on the other hand according to our molecular docking analysis, carvacrol and thymol could interact with BCL2 in BH3 and BH1 positions, they may inhibit BCL2 to lead cancer cells to apoptosis pathway 45 . According to above explanations, since one strategy for cancers therapy is to find molecules that activate the cell death pathways. Hence, we analyzed the interactions of two high-concentration components of ZME with two of BCL2 family members (anti-apoptotic proteins) in the simulated space using molecular docking, and our molecular docking analysis demonstrated that carvacrol and thymol matched the molecules involving in apoptosis; so, they may trigger the apoptosis pathway. Furthermore, some recently studies utilized BCL2 and BCL-xl for docking simulations as anti-apoptotic proteins with several ligands 46,47 and our findings also conform with them. Molecular docking studies and the biological results suggested that ZME can be a promising anticancer agent. In addition, these studies show the plant derivatives beside the alternative drugs and methods such using of nanoparticles, can act as drug complement in clinical therapies, particularly chemotherapeutic treatments 48,49 .
Severe side effects are the most problem of chemotherapy drug, and due to attain a solution to eliminating the problem, researchers attempt to achieve alternatives or complements. Subsequently, the investigation of plant extracts on malignant cells is one of the best elections to induce the programmed cell death. As illustrated in the present study, ZME may be utilized as an alternative or a complement to decrease the effective dose of Dox for pre-B acute lymphoblastic leukemia cells.
Trypan Blue assay. To evaluate the apoptotic effects of ZME and Dox on cell viability, Nalm-6 cells (250 × 10 5 cells/mL) were seeded in 12-well plate and incubated in the presence of the various concentrations of ZME (20,40,80, 100, 200 µg/mL) and Dox (5,10,20,40,80,100 nM) individually for 24, 48, and 72 h. After that, the cell suspension was centrifuged and the cell pellet was suspended in a serum-free complete medium. Next, one part of 0.4% trypan blue (Gibco™ 15250061) and one part of cell suspension was mixed and then allowed mixture to incubate 2 min at room temperature. The total number of unstained (viable) and stained (non-viable) cells was manually counted by Neubauer chamber and light microscope (ECLIPSE E100, Nikon). Finally, the percentage of viable cells was calculated as "Viability (%) = viable cells/viable cells + death cells × 100" 22 .

Determination of combination index and dose reduction index.
To estimate the interaction between ZME and Dox, the combination index (CI) was calculated using CompuSyn Software (ComboSyn, Inc. MTT assay. In vitro screening of the cytotoxicity effect of ZME and Dox towards cancer cell lines was measured using MTT colorimetric assay. The metabolization of thiazolyl blue tetrazolium bromide into formazan crystals by Nalm-6 alive cells was assessed by this test. Hence, 1 × 10 4 Nalm-6 cells were seeded in 96-well plates Figure 4. Fold change gene expression. Graph presents that ZME and DOX upregulate the Bax gene (proapoptotic) and down-regulates Bcl-2 gene (anti-apoptotic). A combination dose of ZME and DOX changes gene expression more than single doses on Nalm-6 cells after 48 h treatment. Bax and Bcl-2 ratio also was shown in this figure and demonstrated the significant effect of ZME and DOX in combination dose. In addition, Bcl-Xl from Bcl-2 family was upregulated by ZME and DOX treatment in Nalm-6 cells after 48 h (*P < 0.05, **P < 0.01, ***P < 0.001, relative to untreated cells). The cells were incubated with 100 µL MTT solution (0.5 mg/mL; (M5655, Sigma) at 37 °C. After 4 h, colored formazan was solubilized by the addition of 150 μL DMSO at each well and optical absorbance was evaluated at 570 nm with an enzyme-linked immunosorbent assay reader. The percentage of metabolic activity of treated cells was calculated relative to untreated cells which were set as negative control. In addition, MTT test was performed for combination dose of ZME/ Dox (100 µg/mL ZME + 10 nM Dox, 100 µg/mL ZME + 20 nM Dox, 200 µg/mL ZME + 10 nM Dox, 200 µg/mL ZME + 20 nM Dox). In addition to untreated cells, Nalm-6 were treated with the highest concentrations of DMSO (were used in our study) as a negative control owing to use it for dissolving ZME (0.01% and 0.1%).
Flowcytometry. The flowcytometry technique was used to assess the effect of ZME and Dox on the induction of early and late apoptosis using annexin V-propidium iodide (PI) staining. Consequently, 4 × 10 5 Nalm-6 Figure 5. Gene expression graph presents that ZME and DOX down-regulates the h-TERT gene and c-Myc gene. A combination dose of ZME and DOX changes gene expression more than single doses on Nalm-6 cells after 48 h treatment. Fold change gene expression graph presents that ZME and DOX up-regulate P21 gene and P53 gene. www.nature.com/scientificreports/ www.nature.com/scientificreports/ cells were seeded into six-well cell culture plates and treated with ZME (100 µg/mL), Dox (10 nM) and combination (100 µg/mL ZME and 10 nM Dox). Then, after 48 h, the cells were collected and they were washed with PBS. Flowcytometry was performed using Annexin-V Apoptosis Detection Kit (Mab Tag, AnxF100PI) and the results were analyzed using the FlowJo.7.6.1 software.
RNA isolation and preparation of cDNA. YTzol Pure RNA (Yekta Tajhiz Azma, YT9066) was used to isolate total RNA from untreated (control) and treated cells with 100 µg/mL ZME, 10 nM Dox and ZME/ Dox combination (100 µg/mL ZME and 10 nM Dox). Quantity of RNA samples was assessed by NanoDrop (Nan-oDrop ND-1000; Thermo Scientific, Wilmington, DE) at A260/A280 ratio. The quality and purity of extracted RNA were illustrated by agarose gel electrophoresis. Reverse transcription (RT) reaction was carried out according to the manufacturer instructions using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific Fermentas, K1622).

Quantitative Real-time PCR.
Changes in mRNA expression of desired genes were surveyed by real-time PCR. Quantitative real-time PCR was performed by 10 μL containing Real Q Plus 2 × Master Mix Green (Amplicon, Denmark, A325402), 1.5 μL of the cDNA product, 1 μL of forward and reverse primers (10 pmol of each other), and 7.5 μL of nuclease-free water. Thermal cycling conditions included an initial activation step at 95 °C for 15 min followed by 40 cycles, a denaturation step at 95 °C for 15 s and a combined annealing/elongation step at 60 °C for 60 s. The reaction took place in the RotorGene® Q Real-time PCR System (Qiagen, USA). A melting curve analysis was performed to verify the specificity of the products. The fold change was measured relative to the control and calculated after adjusting for the B-actin reference gene using Ct (2 −ΔΔCT ) method. Nucleotide sequences of the primers used for real-time RT-PCR listed in Table 3. ZME effect on normal cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor using density gradient centrifugation using Ficoll-Hypaque density gradient (Lymphodex, Germany). Isolated cells were washed two times by PBS. Thus, the pellet was resuspended in 1 mL complete media (containing RPMI-1640 with 2 mM l-glutamine, 10% FBS and 1% antibiotic) and cultured in 8 mL complete media at the same condition used for Nalm-6 cells. These cells were treated with ZME (100 and 200 µg/mL) and combination (ZME: 100 µg/mL with Dox: 10 nM) and incubated at 37 °C so as to assess metabolic activity after 48 h using MTT. PBMCs, as well, were treated with 100 µg/mL ZME and combination dose (100 µg/mL ZME and 10 nM Dox) and measure the apoptosis rate after 48 h by flowcytometry.
Molecular docking. The 3D structure of antiapoptotic proteins such as BCL2 (PDB ID: 2W3L) 25 and BCLxl (PDB ID: 2YXJ) 26 were obtained from the RCSB protein data bank (https:// www. rcsb. org). In addition, the ligands of these complexes were eliminated using the software MOE 2019.102 (Molecular Operating Environment) to prepare the macromolecules for docking stimulation. Thymol (CID:6989), carvacrol (CID:10364) structures were retrieved from PubChem compound database (https:// pubch em. ncbi. nlm. nih. gov/). MOE was used to compute and find the active sites of BCL2 and BCL-xl for ligands binding. Here, AutoDock Tools 4.2 and MVD software (Molegro Virtual Docker 6.0.1) have been applied for molecular docking. The active sites were input and the grid box dimension has been adjusted to optimized size surrounding the active site of the protein in order to ensure the free rotation of the ligands in the inner side of the grid. The docking run numbers have been estimated to be 100. The resulting poses have been chosen according to the corresponding binding energy. Using LigPlot analysis, the interactions of ligands and involved amino acids of proteins were analyzed, and the results depict in 2D interaction between ligands and macromolecules. Ultimately, the 3D shapes of the final interaction were drawn using the software Chimera 1.12 to clarify the interactions.